Method and system for sampling material from cells

ABSTRACT

Methods, systems, and devices for sampling/isolating material from cells. An exemplary system may comprise a chip including an electrode array of sampling electrodes arranged along a surface of the chip. A cell-receiving area may be located adjacent the surface of the chip. The system also may comprise a tag array of tags supported by the chip and aligned with the electrode array. Each tag of the tag array may include an identifier that is unique to the tag within the tag array. Each tag may be configured to bind nucleic acids, or a capturing agent distinct from the tag may be aligned with each sampling electrode of the electrode array to capture a protein or other analyte of interest. The system further may comprise a control circuit configured to apply an individually controllable voltage to each sampling electrode of the electrode array and measure an electrical property of the sampling electrode.

CROSS-REFERENCE TO PRIORITY APPLICATION

This application is a continuation of U.S. patent application Ser. No.17/105,333, filed Nov. 25, 2020, now U.S. Pat. No. 11,542,541, which, inturn, is based upon and claims the benefit under 35 U.S.C. § 119(e) ofU.S. Provisional Patent Application Ser. No. 62/940,866, filed Nov. 26,2019. Each of these applications is incorporated herein by reference inits entirety for all purposes.

INTRODUCTION

The transcriptome is the complete set of RNA transcripts, and thequantity of each RNA transcript, present in a cell and/or expressed froma genome under specific conditions and/or at a particular developmentalstage. The study of the transcriptome is called transcriptomics and isconcerned not just with cataloging RNA transcripts for a given cell butalso quantifying changing expression levels in response to hormones,drugs, environmental factors, differentiation, development, or the like.

RNA sequencing provides a high-throughput approach to catalog thetranscriptome of a cell. In this approach, RNA from a cell serves as atemplate for synthesis of complementary DNA (cDNA). The cDNA is thenprepared for sequencing, such as by ligation to linkers andamplification.

Spatial transcriptomics is the study of the transcriptome of a genome intwo or more dimensions, such as across a section of tissue. A techniquefor parallel acquisition of two-dimensional (2D) RNA sequencinginformation has been developed. In the technique, a tissue section islaid upon a slide carrying an arrangement of beads. RNA released fromcells of the tissue section diffuses to the beads and serves as atemplate for synthesis of distinguishably tagged cDNA at each bead.Tagged cDNAs from different beads then are pooled and sequenced. cDNAshaving the same tag are assumed to represent RNA molecules thatoriginated from the same region or cell of the tissue section. However,the technique produces libraries of variable quality and complexity. Newmethods and systems for sampling nucleic acids from cells forsequencing, such as 2D RNA sequencing, are needed.

SUMMARY

The present disclosure provides methods, systems, and devices forsampling/isolating material, such as nucleic acids or a particularprotein, from cells. An exemplary system may comprise a chip includingan electrode array of sampling electrodes arranged along a surface ofthe chip. A cell-receiving area may be located adjacent the surface ofthe chip. The system also may comprise a tag array of tags supported bythe chip and aligned with the electrode array. Each tag of the tag arraymay include an identifier that is unique to the tag within the tagarray. Each tag may be configured to bind nucleic acids, or a capturingagent distinct from the tag may be aligned with each sampling electrodeof the electrode array to capture a protein or other analyte ofinterest. The system further may comprise a control circuit configuredto apply an individually controllable voltage to each sampling electrodeof the electrode array and measure an electrical property of thesampling electrode.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of an exemplary system for sampling materialfrom cells using an array of sampling electrodes.

FIG. 2 is a schematic side view of an exemplary isolation device toprovide the array of sampling electrodes for the system of FIG. 1 .

FIG. 3 is a flowchart listing exemplary steps that may be performed inan exemplary method of sampling material from cells using an array ofsampling electrodes.

FIG. 4 is a pair of graphs showing illustrative voltage that may beapplied to a sampling electrode of an array of sampling electrodes overa time period during performance of the method of FIG. 3 , andillustrative electric current that may be measured from the samplingelectrode for the same time period.

FIG. 5 is a graph plotting illustrative electric current that may bemeasured from ten sampling electrodes of an array of sampling electrodesduring performance of the method of FIG. 3 .

FIG. 6 is an exemplary capture prediction map that may be generatedduring performance of the method of FIG. 3 using the data plotted in thegraph of FIG. 5 .

FIG. 7 is a fragmentary, top plan view of an exemplary chip for themethods, systems, and devices of the present disclosure, where the chiphas an array of sampling electrodes each located at the bottom of adifferent well.

FIG. 8 is a fragmentary, top plan view of another exemplary chip for themethods, systems, and devices of the present disclosure, where the chipis similar to that of FIG. 7 , except for the addition of a single guardelectrode encircling each of the different wells.

FIG. 9A is a fragmentary, top plan view of yet another exemplary chipfor the methods, systems, and devices of the present disclosure, wherethe chip is similar to that of FIG. 8 , except that the chip has aplurality of guard electrodes collectively encircling each of thedifferent wells and each individually encircling only one of thedifferent wells.

FIG. 9B is a fragmentary, top plan view of still another exemplary chipfor the methods, systems, and devices of the present disclosure, wherethe chip is similar to that of FIG. 9A, except that the samplingelectrodes, wells, and guard electrodes are arranged in a hexagonalarray instead of a rectangular array.

FIG. 10 is a fragmentary, sectional view of an exemplary isolationdevice including the chip of FIG. 8 and a removable cover providing ashared counter electrode, taken generally along line 10-10 of FIG. 8through one of the wells and a corresponding sampling electrode.

FIG. 11 is a circuit diagram of an exemplary circuit portion for thecontrol circuit of the sampling system of FIG. 1 , wherein the circuitportion may be utilized to set the voltage bias of the counter electrodeof FIGS. 1 and 10 or to set the voltage bias of the guard electrode ofFIG. 10 .

FIG. 12 is a circuit diagram of an exemplary circuit portion for thecontrol circuit of the sampling system of FIG. 1 , where the circuitportion may be utilized to set the voltage bias of the guard electrodesof FIGS. 9A and 9B.

FIG. 13 is a circuit diagram of an exemplary circuit portion for thecontrol circuit of the sampling system of FIG. 1 , where the circuitportion may be utilized to set the voltage bias and sense the current ofsampling electrodes of the sampling system.

FIG. 14 is a circuit diagram of another exemplary circuit portion forthe control circuit of the sampling system of FIG. 1 , where the circuitportion may be utilized to set the voltage bias and sense the current ofsampling electrodes of the sampling system.

FIG. 15 is an isometric view of an exemplary isolation device for thesampling system of FIG. 1 , with a cover of the isolation deviceremoved, and with the isolation device having only a small number ofwells and corresponding sampling electrodes for clarity.

FIG. 16 is a top plan view of the isolation device of FIG. 15 .

FIG. 17 is a fragmentary sectional view of the isolation device of FIG.15 , taken generally along line 17-17 of FIG. 16 is the presence of thecover.

FIG. 18 is fragmentary sectional view of another exemplary isolationdevice, taken as in FIG. 17 , but with the well containing a removablebead to which a tag of a tag array is attached.

FIG. 19 is a flowchart listing exemplary steps of an exemplary method ofsampling nucleic acids from cells of a tissue section.

FIG. 20 is a flowchart listing exemplary steps of an exemplarylysis/electrophoresis routine that may be performed within the method ofFIG. 19 .

FIG. 21 is a schematic fragmentary sectional view of an exemplaryconfiguration of an exemplary isolation device produced after performingthe first five steps of the method of FIG. 19 .

FIGS. 22-27 are schematic fragmentary sectional views of exemplaryconfigurations that may be produced with the isolation device of FIG. 21during performance of the method of FIG. 19 , except with an electrolyteliquid rather than an electrolyte gel placed on top of the tissuesection.

FIG. 28 is an exploded fragmentary isometric view of an exemplaryisolation device for sampling nucleic acids from isolated cells insteadof cells of a tissue section, where the isolation device has a series ofcapillary tubes each communicating laterally with a different row ofwells of the isolation device.

FIGS. 29-34 are schematic fragmentary sectional views of the isolationdevice of FIG. 28 , taken generally along line 29-29 of FIG. 28 andillustrating a series of exemplary configurations that may be producedwith the isolation device of FIG. 28 during performance of aspects ofthe sampling method of FIG. 3 .

FIG. 35 is an isometric view of an exemplary channel-forming member thatcan be bonded to the top surface of a chip of the isolation device ofFIG. 28 , to replace the series of capillary tubes of FIG. 28 .

FIG. 36 is an isometric view of a chamber-forming member than can bebonded to the top surface of a chip of the isolation device of FIG. 28 ,to replace the series of capillary tubes.

FIGS. 37 and 38 are schematic fragmentary sectional views of anembodiment of the collection device of FIG. 3 , taken through a well andcorresponding sampling electrode and illustrating an exemplaryconfiguration of electrode-associated reagents for capturing and taggingDNA sequences released from an overlying cell.

DETAILED DESCRIPTION

The present disclosure provides methods, systems, and devices forsampling/isolating material (e.g., nucleic acids, such as RNA, or aprotein or other analyte) from cells. An exemplary system may comprise achip including an electrode array of sampling electrodes arranged alonga surface of the chip, and optionally located at the bottom of acorresponding array of wells. A cell-receiving area may be locatedadjacent the surface of the chip. In some examples, the cell-receivingarea may be configured to receive a tissue section including cells. Thesystem also may comprise a tag array composed of tags, such as primers,supported by the chip and aligned with the electrode array. Each tag ofthe tag array may include an identifier that is unique to the tagrelative to each other tag of the tag array. Each tag may be configuredto bind nucleic acids, or a capturing agent distinct from the tag may bealigned with each sampling electrode of the electrode array to capture aprotein or other analyte of interest. For example, each tag may beconfigured to hybridize with a poly(A) tail of messenger RNA molecules.The system further may comprise a control circuit configured to apply anindividually controllable voltage to each sampling electrode of theelectrode array and measure an electrical property of the samplingelectrode. In some examples, the control circuit may be configured toapply a lysis voltage to the sampling electrode if the electricalproperty measured for the sampling electrode meets one or more criteria.

An exemplary method of sampling material from cells is disclosed. In themethod, a plurality of cells may be received on a surface of a chip. Thechip may include an electrode array of sampling electrodes arrangedalong the surface. An electrical property may be measured for eachsampling electrode of the electrode array. A lysis voltage may beapplied to the sampling electrode if the measured electrical propertyfor the sampling electrode meets one or more criteria. Anelectrophoresis voltage may be applied to the sampling electrode, if thelysis voltage was applied to the sampling electrode, to drive nucleicacids (or other cell material of interest, such as a particular proteinor other analyte), if any, that were released from one of the cells byapplying the lysis voltage, toward the sampling electrode.

An exemplary method of sampling nucleic acid material from a tissuesection is disclosed. In the method, a chip may be selected. The chipmay include an electrode array of sampling electrodes arranged along asurface of the chip. The chip may support a primer array aligned withthe electrode array. The primer array may be composed of primers, witheach primer being configured to hybridize to a poly(A) tail of RNA andincluding an identifier that is unique to the primer within the primerarray. The tissue section may be received on the surface of the chip. Anelectrical property may be measured for each sampling electrode of theelectrode array. A lysis voltage may be applied to the samplingelectrode if the measured electrical property for the sampling electrodemeets one or more criteria. RNA molecules from the tissue section may becaptured with each primer of a plurality of different primers of theprimer array.

The present disclosure offers various improvements and advantages for 2DRNA sequencing to determine a 2D transcriptome profile of a tissuesection, including any combination of the following. Known methods ofpreparing 2D RNA sequencing libraries rely on passive diffusion of RNAto beads carrying primers. However, RNA migration to the beads can bevery inefficient, which causes inaccurate representation of RNA species,and the absence of rare RNA species, in the resulting library. Incontrast, systems and methods of the present disclosure utilizeelectrophoresis to actively drive nucleic acids toward samplingelectrodes. Actively driving nucleic acids facilitates capture and mayresult in a much higher yield of RNA captured by a respective primerlocated over each sampling electrode and much less lateral migration ofRNA. In addition, known methods of preparing libraries for 2D RNAsequencing do not control release of RNA from individual cells of atissue section. Instead, RNA is released from the tissue section bytreating the entire tissue section with a reagent(s) to effect chemicallysis. As a result, RNA from different cells can migrate to the samebead, which destroys or at least degrades single-cell resolution of theresulting library. In contrast, systems and methods of the presentdisclosure use sampling electrodes to actively rupture membranes ofcells by electrical lysis. This electrical lysis can be controlledelectronically, to precisely control when and where lysis occurs acrossa tissue section, in some cases at a single-cell resolution. Moreover,this electrical lysis can be followed by electrophoresis to activelydrive released nucleic acids to a primer or other capturing agentlocated over a sampling electrode. Accordingly, RNA can be controllablyreleased with precision from selected cells of the tissue section in aspatially restricted manner and driven toward an aligned samplingelectrode. In combination, these procedures may produce more efficientcapture of RNA, and less lateral migration of RNA and mis-tagging ofcDNA that degrades the quality of the resulting library. Furthermore,known methods of preparing libraries for 2D RNA sequencing do notprovide any assessment of RNA release or capture, or library quality,without going through all the trouble and expense of preparing a libraryand acquiring sequence data from the library. In contrast, methods andsystems of the present disclosure can predict, optionally in real time,where RNA has been captured with respect to an electrode array ofsampling electrodes. The prediction may be based on electricalmeasurements and/or application of a lysis potential and/or anelectrophoresis potential to particular sampling electrodes.

The methods and systems may utilize or include a chip having an array ofwells that are aligned with an array of sampling electrodes. Eachsampling electrode may be located at the bottom of a different well. Thediameter of each well can be smaller or larger than the diameter ofcells that are disposed on the chip. Accordingly, each well can receivenucleic acids from a single cell of a tissue section, if the diameter ofthe well is smaller than the diameter of the cells, or from multiplecells of a tissue section, if the diameter of the well is larger thanthe diameter of the cells. The presence of wells can offer variousbenefits. First, wells allow sampling electrodes, tags, capturingagents, and/or the like, to be located in and/or at the bottom of thewells, with an offset below the mouth of the well. With this offset, thelip of each well can be formed of a dielectric material. When a cell(s)covers the mouth of the well and moves into very close proximity orcontact with the lip of well, the cell may be advantageously positionedfor electrical lysis. Electric current may be forced to flow in a narrowgap, if any, between the cell and the lip of the well, because thecell's membrane and the lip are electrically insulating. This results ina high current density at the cell's membrane when a lysis voltage isapplied, which can rupture the cell. Moreover, the positioning of a cellover the mouth of the well and very close to the well's lip can bedetected as an increase in resistance to current flow between a samplingelectrode at the bottom of the well and a counter electrode.Accordingly, by measuring an electrical property of the samplingelectrode that reflects the resistance, an optimal timing for electricallysis and a prediction of the success of the electrical lysis can bedetermined. Using this approach, the methods and systems canintelligently apply a lysis voltage followed by an electrophoresisvoltage and an optional reverse voltage, to selected samplingelectrodes.

The electronic component of the method and system provides advantages.First, rupturing a cell membrane electrically facilitates RNA capture.Second, the ability to predict where cells have been lysed offers areal-time assessment of the efficiency and location of captured nucleicacids. This information allows a user to evaluate the prospectivetwo-dimensional quality of a library before it is constructed or usedfor sequencing. Recording the activities of sampling electrodes allowsreal-time display or post-sampling construction of a saturation map(i.e., a map of the sampling electrodes showing where RNA capture ispredicted to have occurred). The saturation map allows the user to makeknowledge-based decisions about the next steps, for example, whether toproceed with sequencing (e.g., based on quality, tissue surfacecoverage, etc.). Third, if a respective well is aligned with eachsampling electrode, electric potential applied to the sampling electrodequickly dissipates lateral to the top of the well, which allowsprecise/specific collection of RNA from a cell(s) aligned with the well.Fourth, the method and system may use or include a chip having anintegrated circuit (digital) connected to sampling electrodes (analog),where the sampling electrodes are positioned for contact with anelectrolyte placed on the chip. The chip may, for example, have 1,000 to100,000 sampling electrodes. Each sampling electrode may be located in aseparate well, which may, for example, be 1-70 micrometers in diameterand 1-70 micrometers deep. Each well may contain a primer unique to thatwell, relative to each other well, and configured to capture RNA. Theprimer may include a unique identifier, such as a barcode sequence. Eachelectrode can be controlled individually and may measure acurrent/potential, which may be recorded for further analysis.

Further aspects of the present disclosure are described in the followingsections: (I) definitions, (II) system, device, and method overview,(III) examples, and (IV) selected aspects.

I. Definitions

Technical terms used in this disclosure have meanings that are commonlyrecognized by those skilled in the art. However, the following terms maybe further defined as follows.

Aligned—arranged along or centered on the same axis, such as an axisorthogonal to a planar surface/side of a chip or a central axis of awell of the chip.

Array—an arrangement of related elements (e.g., sampling electrodes,wells, tags, primers, identifiers, etc.), optionally a systematicarrangement, such as in rows and/or columns. The arrangement may includeany suitable number of the elements, such as at least 10, 100, 1,000,10,000, or 100,000, among others. An array may be an electrode arraycomposed of an array of sampling electrodes, a well array composed of anarray of wells, a tag array composed of an array of structurallydifferent tags (e.g., different primers), or the like. Any arraydisclosed herein may be a planar array having its elements arrangedalong a plane, such as a plane defined by a surface of a chip (e.g., atop side and/or planar side of the chip). Two or more different arrays,such as an electrode array and a well array, or an electrode array and atag array, may be parallel to the same plane. Any array disclosed hereinmay be a subarray of a larger array of the same type of elements. Forexample, a chip may include an electrode array composed of 1,000sampling electrodes, and the electrode array may be a subarray within alarger array of more than 1,000 sampling electrodes on the chip. Twoarrays are aligned with one another if each element of one of the arraysis aligned with a different element of the other array.

Capturing agent—a molecule, molecular region, or molecular complex thatis immobilized and binds nucleic acids, such as by hybridization (i.e.,base pairing), and/or attaches nucleic acids to an immobilized tag. Thecapturing agent may be immobilized by connection to a chip or a bead,among others. A capturing agent may be provided by a tag (e.g., by aregion of the tag including a series of nucleotides), a protein(s), orthe like. A capturing agent provided by a tag also may be called acapturing sequence. In some examples, the capturing agent is a specificbinding partner, such as an antibody, that binds to a protein or otheranalyte of interest to be captured from lysed cells.

Cell—the basic structural, functional, and biological unit of a livingorganism. Exemplary cells include stem cells, established cells (e.g.,cell lines), primary cells, cells of a tissue, transfected cells, cellsfrom a clinical sample (e.g., a blood sample, a fluid aspirate, a tissuesection, etc.), and/or the like. The cells of the present disclosure maybe tissue cells or isolated cells, among others. The term “isolatedcells” refers to single cells and/or clusters of cells that are notconnected to one another.

Chip—an article including an integrated circuit. A chip may be referredto as being flat or a plate, which means that the chip has a length andwidth that are much greater than the chip's thickness, such as at leastten times the thickness. The chip may include a substrate, which is alayer or substratum below one or more other layers of the chip. Thesubstrate may be formed of a semiconductor, such as silicon, galliumarsenide, gallium nitride, silicon carbide, or the like. The chip has a“top side” that can be oriented parallel to a horizontal plane andfacing up, whether or not the chip is used for sampling nucleic acids inthis orientation. The chip, and particularly the top side thereof, mayhave any suitable size and shape to match the size, shape, and/or typeof target tissue/cells from which nucleic acids will be sampled.

Complementary—related by the rules of base pairing. A first nucleicacid, or region thereof, is “complementary” to a second nucleic acid ifthe first nucleic acid or region is capable of hybridizing with thesecond nucleic acid in an antiparallel fashion by forming a consecutiveor nearly consecutive (uninterrupted) series of base pairs. The firstnucleic acid (or region thereof) is termed “perfectly complementary” tothe second nucleic acid if hybridization of the first nucleic acid (orregion thereof) to the second nucleic acid forms a consecutive series ofbase pairs using every nucleotide of the first nucleic acid or regionthereof. A “complement” of a first nucleic acid is a second nucleic acidthat is perfectly complementary to the first nucleic acid for at leastten consecutive nucleotides. The “complementarity” between a firstnucleic acid (or region thereof) and a second nucleic acid (or regionthereof) refers to the number or percentage of base pairs that can beformed when the first nucleic acid (or region thereof) is optimallyaligned for hybridization in an antiparallel fashion with the secondnucleic acid (or region thereof). A first nucleic acid or region thereofthat is complementary to a second nucleic acid or region thereofgenerally has a complementarity of at least 80% or 90%.

Control circuit—electronic circuitry configured to control and senseelectrical properties of electrodes and including any number of digitaland analog circuits, and optionally including a processor to performoperations on a data stream. A control circuit may be configured tocommunicate with a user and to output data reporting the current statusor final results of a sampling run performed with the systems anddevices of the present disclosure.

Degenerate—having a mix of sequences or nucleotide identities. A“degenerate primer” or a “degenerate tag” refers to a mix of primeroligonucleotides or tag oligonucleotides of related but not identicalsequence. The aligned sequences of the primer oligonucleotides or tagoligonucleotides collectively define a “consensus sequence” for thedegenerate primer or degenerate tag, which indicates the identity of thesingle nucleotide, or two or more different nucleotides, present at eachnucleotide position along the degenerate primer or tag. The degeneracyof the degenerate primer or tag refers to the number of different primeror tag oligonucleotide sequences constituting the degenerate primer ortag, and the degeneracy of a nucleotide position of the degenerateprimer or tag indicates how many different nucleotides are present atthat position in the mix. The degeneracy of a given nucleotide positionof a degenerate primer or tag may be 1, 2, 3, or 4, among others. Each“N” in the consensus sequence of a degenerate primer or tag increasesits degeneracy by a factor of four.

Electrode—a conductor for contacting an electrolyte. The conductor maybe formed of any suitable electrically conductive material, such as anoble metal or a noble metal alloy (e.g., gold, platinum, rhodium,iridium, palladium, ruthenium, osmium, or a combination thereof). Anelectrode may, for example, be a sampling electrode, a guard electrode,or a counter electrode. The term “sampling electrode” refers to anelectrode toward which nucleic acids are attracted, such as for captureand isolation. The term “guard electrode” refers to an electrode locatedintermediate two or more sampling electrodes and configured to increaseelectrical isolation of the sampling electrodes from one another. Theterm “counter electrode” refers to an electrode that can completecircuits with sampling electrodes and/or one or more guard electrodesand allows a voltage to be applied to the sampling electrodes and/orguard electrode(s). Accordingly, application of a voltage to anelectrode, such as a sampling electrode, as used herein, means that thevoltage is applied between the electrode and a counter electrode. Thephrase “at a sampling electrode” means in close proximity to thesampling electrode, such as located in a well that is aligned with thesampling electrode, but not necessarily in contact with the samplingelectrode. The phrase “at a sampling electrode” can mean located closestto the sampling electrode relative to each other sampling electrode ofan electrode array, and/or within a distance of 50, 20, 10, or 5micrometers of the sampling electrode.

Electrolyte—an electrically conductive medium including free ions insolution. An electrolyte may be a liquid (i.e., an electrolyte liquid)or a gel (i.e., an electrolyte gel). An exemplary electrolyte of thepresent disclosure includes an aqueous solution of free ions.

Lysis—disintegration of the membrane and/or wall of a cell(s). The verb“lyse” means undergoing or causing to undergo lysis.

Messenger RNA—RNA containing a poly(A) tail and/or that conveys geneticinformation to a ribosome.

Nucleic acid—a polymer of any length composed of naturally-occurringnucleotides (e.g., where the polymer is DNA or RNA), or a substanceproduced synthetically that can hybridize with DNA or RNA in asequence-specific manner analogous to that of two naturally occurringnucleic acids, e.g., can participate in Watson-Crick base pairinginteractions. A nucleic acid may be composed of any suitable number ofnucleotides, such as at least about 5, 10, 100, or 1000, among others.Generally, the length of a nucleic acid corresponds to its source, withsynthetic nucleic acids (e.g., oligonucleotides) typically beingshorter, and biologically/enzymatically generated nucleic acids (e.g.,genomic fragments) typically being longer.

A nucleic acid may have a natural or artificial structure, or acombination thereof. Nucleic acids with a natural structure, namely,deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), generally have abackbone of alternating pentose sugar groups and phosphate groups. Eachpentose group is linked to a nucleobase (e.g., a purine (such as adenine(A) or guanine (G)) or a pyrimidine (such as cytosine (C), thymine (T),or uracil (U))). Nucleic acids with an artificial structure are analogsof natural nucleic acids and may, for example, be created by changes tothe pentose and/or phosphate groups of the natural backbone and/or toone or more nucleobases. Exemplary artificial nucleic acids includeglycol nucleic acids (GNAs), peptide nucleic acids (PNAs), lockednucleic acids (LNAs), threose nucleic acids (TNAs), xeno nucleic acids(XNA), and the like.

The sequence of a nucleic acid is defined by the order in whichnucleobases are arranged along the backbone. This sequence generallydetermines the ability of the nucleic acid to hybridize with anothernucleic acid by hydrogen bonding. In particular, adenine pairs withthymine (or uracil) and guanine pairs with cytosine.

Oligonucleotide—a relatively short and/or chemically synthesized nucleicacid. The length of an oligonucleotide may, for example, be 3 to 1000nucleotides, among others.

Primer—an oligonucleotide capable of serving as a point of initiation oftemplate-directed nucleic acid synthesis under appropriate reactionconditions (e.g., in the presence of a template to which the primerhybridizes, nucleoside triphosphates, and an enzyme to catalyzepolymerization (such as a DNA or RNA polymerase or a reversetranscriptase), in an appropriate buffer and at a suitable temperature).The primer may have any suitable length, such as 5 to 500 nucleotides,among others. The term “primer binding site” refers to a portion of atemplate to which a primer anneals. The full sequence of the primer neednot be perfectly complementary to the primer binding site, justsufficiently complementary to anneal under the conditions of thereaction. Accordingly, the primer may have a 3′-end region that iscomplementary to the primer binding site, and a 5′-end region that isnot complementary to the primer binding site.

Taq—an oligonucleotide including an identifier, which may be composed ofany suitable number of nucleotides, such as at least 4, 5, 6, or morenucleotides. The identifier may be described as a unique molecularidentifier or molecular barcode. A tag array may be composed of tagseach including a different identifier that is unique within the tagarray, that is, with respect each other tag of the tag array. Thesequence of the different (unique) identifier present at each positionof the tag array may be known, such that each identifier sequencerepresents a known spatial position within the array. Except fordifferent identifier sequences, the sequences of the tags may beidentical to one another otherwise. In some examples, each tag also mayinclude a capturing agent that hybridizes to nucleic acids, such asmessenger RNA molecules, via a capturing sequence of the tag. Thecapturing sequence may, for example, be configured to hybridize to thepoly(A) tail of messenger RNA. In other examples, the capturing agentand the tag may be discrete relative to one another. The uniqueidentifier sequence of each tag may be used, when analyzing sequencedata from a resulting library, to identify the particular samplingelectrode and/or well in the electrode array and/or well array fromwhich each sequenced library member originated.

Template—a nucleic acid that serves as a pattern for synthesis of acomplementary strand. The template may provide a primer binding site fora primer, which is extended by sequential addition of complementarynucleotides according to the pattern.

Tissue—an aggregate of cells from a multicellular organism or created invitro. Tissue may, for example, form a structural material with one ormore specific functions or represent abnormal growth (such as cancer).The term “tissue” includes a single type of tissue, such as muscle,nervous, connective, epithelial, tumor, or the like, or two or moretypes of tissue that are connected to one another to create a morecomplex structure, such as an organ or portion thereof. A “tissuesection” refers to a slice of tissue. The tissue section may have anysuitable thickness, such as less than (or greater than) about 100, 50,20, or 10 micrometers, among others, and/or less than (or greater than)about 5, 3, or 2 times the average diameter of cells in the tissuesection. The tissue section may be obtained from any suitable organ ortissue type, and may be prepared as a frozen section, a fixed section, afresh section, and/or the like.

Well—a depression or recess of any suitable size and shape. The wells ofa well array, as disclosed herein, may have a diameter of less than 100,50, 25, 10, or 5 micrometers, and/or a diameter greater than 1, 2, or 5micrometers, among others. The diameter may, for example, be 1-100,1-70, or 1-50 micrometers, or the like. The wells may have a depth ofless than 100, 50, 25, 10, or 5 micrometers, and/or a depth greater than1, 2, or 5 micrometers, among others. The depth may, for example, be1-100, 1-70, or 1-50 micrometers, or the like. The diameter of the wellsmay be greater than, less than, or about the same as the depth.Accordingly, the wells may be shallow recesses, deep holes, or somethingintermediate these extremes. Each well may have any suitable shape, suchas cylindrical, conical, rectangular, or the like.

II. System, Device, and Method Overview

This section provides an overview of the sampling systems, devices, andmethods disclosed herein; see FIGS. 1-3 .

FIG. 1 shows an exemplary sampling system 50 for sampling nucleic acidsfrom cells 52 using an electrode array 54 of sampling electrodes 56,which are provided by a chip 58. Only three illustrative samplingelectrodes 56 a, 56 b, and 56 c are shown in FIG. 1 for clarity.However, chip 58 may include any suitable number of sampling electrodes56, such as at least 100 or 1,000 sampling electrodes, as indicated byan ellipsis between illustrative sampling electrodes 56 b and 56 c.Sampling system 50 is shown and described herein with electrode array 54in a horizontal configuration and facing up, but electrode array 54 maybe used in any suitable orientation.

Chip 58 may define a well array 60 aligned with electrode array 54. Wellarray 60 is composed of wells 62, including illustrative wells 62 a, 62b, and 62 c. Each sampling electrode 56, such as illustrative samplingelectrodes 56 a, 56 b, and 56 c, may be aligned with a different well 62of well array 60, such as illustrative wells 62 a, 62 b, and 62 c,respectively, and may be located in the different well, such as at thebottom thereof. Accordingly, the top surface of each sampling electrode56 may be recessed with respect to a surrounding dielectric top surfaceof chip 58. In other examples, wells 62 may be omitted and the topsurface of each sampling electrode 56 may be flush or elevated withrespect to a dielectric top surface of the chip.

Chip 58 may support a tag array 64 of tags 66, including illustrativetags 66 a, 66 b, and 66 c. Tags 66 each may have a different nucleotidesequence. Tag array 64 may be aligned with electrode array 54 and wellarray 60, with copies of a different tag 66 of tag array 64 located overeach sampling electrode 56, such as vertically aligned with theelectrode and the corresponding well. For example, in FIG. 1 ,illustrative tags 66 a, 66 b, and 66 c are vertically aligned withillustrative electrodes 56 a, 56 b, and 56 c, respectively, andvertically aligned with illustrative wells 62 a, 62 b, and 62 c,respectively. Tags 66 may be located in corresponding wells 62 and maybe primers.

Each tag 66 of tag array 64 may include a different identifier 68, suchas illustrative identifiers 68 a, 68 b, and 68 c (also called uniquemolecular identifiers (UMIs)). Tags 66 may differ in nucleotide sequencefrom one another only at identifiers 68, which may be created bysubsequences in the tags. Each identifier ultimately may provide arecord of the particular sampling electrode and/or well within theelectrode array and/or well array from which each corresponding librarymember originated.

Copies of a capturing agent 70 may be aligned with each samplingelectrode 56 and well 62. Capturing agent 70 may be provided by tags 66.For example, in FIG. 1 , each of illustrative tags 66 a, 66 b, and 66 cincludes the same capturing agent 70, which may be formed by a distinctsubsequence within each tag relative to identifiers 68 a, 68 b, and 68c.

Sampling system 50 may release cell material, such as nucleic acids,from individual cells 52 by electrical lysis. This lysis may be inducedby energizing selected sampling electrodes 56 of electrode array 54 viaa control circuit 72. The control circuit 72 may be in electricalcontact with each sampling electrode 56, indicated at 73, and inelectrical contact with one or more counter electrodes(s) 74, indicatedat 76. Nucleic acids may be released from one or more cells of cells 52that are located vertically above a given energized sampling electrode56 and/or the corresponding well 62, and electrophoresed toward thegiven sampling electrode 56. Electrophoresis of the released nucleicacids may be driven by further energization of the given samplingelectrode 56 via control circuit 72, to allow capture by copies ofcapturing agent 70 located over the given sampling electrode 56. Thecaptured nucleic acids, or fragments thereof, may be tagged by theparticular tag 66 located over the given sampling electrode 56, or maytemplate the synthesis of complements of the captured nucleic acids,which may be tagged by the particular identifier 68, as explainedfurther below.

Sampling system 50 includes an electrolytic assembly 78 formed bycounter electrode(s) 74, an electrolyte 80 (gel and/or liquid), cells52, electrode array 54, and control circuit 72. Electrolyte 80 may belocated in an electrolyte-receiving space 81 adjacent counterelectrode(s) 74 (also see FIG. 2 ). The electrolyte may be positionedunder and in contact with counter electrode(s) 74, indicated at 82.Electrolyte 80 also may be located adjacent, indicated at 83, acell-receiving area 84 for cells 52, which in turn may be adjacent a topsurface of chip 58. Electrolyte 80 may be located under and over cells52, with electrolyte 80 also present in wells 62.

Counter electrode(s) 74 is spaced from electrode array 54, optionallylocated vertically above electrode array 54, and provides a counterelectrode for each sampling electrode 56 of electrode array 54. In someexamples, counter electrode(s) 74 may be described as a topelectrode(s). Counter electrode(s) 74 may, for example, be formed by asingle conductive plate or a plurality of separate conductive plates.The horizontal area occupied by counter electrode(s) 74 may be at leastas large as the horizontal extent (length times width) occupiedcollectively by electrode array 54, and/or each sampling electrode 56may be vertically aligned with counter electrode(s) 74.

During operation of system 50, the electric potential of counterelectrode(s) 74 may be held constant by control circuit 72, to providean electric potential reference, while the electric potentials ofsampling electrodes 56 are varied, to apply voltages individually toelectrodes 56. Control circuit 72 may be configured to switchably andindividually create a negative potential difference or a positivepotential difference between any given sampling electrode 56 and counterelectrode(s) 74. In other words, counter electrode(s) 74 can switchbetween serving as an anode (positive potential) and a cathode (negativepotential) and with respect to any given sampling electrode 56.

Sample system 50 also may include a user interface 88 to permit a userto communicate with control circuit 72. User interface 88 may includeany suitable combination of one or more input devices (e.g., a keyboard,a mouse, a keypad, a touch screen, and/or the like) and one or moreoutput devices (e.g., a display, a touch screen, a printer, a speaker,and/or the like). The user interface may output data to the user duringa sampling run (e.g., reported and updated in real time), and/or afterthe end of the sampling run. The data may report a number, a percentage,and/or a map of sampling electrodes 56 vertically over which cell lysisand/or capture of nucleic acids is predicted to have occurred.

FIG. 2 shows a schematic side view of an exemplary isolation device 90to provide at least part of sampling system 50 of FIG. 1 . Isolationdevice 90 includes chip 58 having electrode array 54 (of samplingelectrodes) and well array 60 as described above. Chip 58 may or may notbe removable from isolation device 90. In some examples, chip 58 may bedisposable (single-use) and the rest of isolation device 90 may bereusable.

Chip 58 also may include one or more guard electrodes 92 that functionto electrically isolate sampling electrodes 56 from one another. Thehorizontal position of guard electrode(s) 92 is not shown accurately inFIG. 2 , as guard electrode(s) 92 is typically vertically aligned withthe area bounded by a polygon circumscribing electrode array 54. SeeExample 2 for exemplary guard electrodes that may be suitable for chip58.

Chip 58 further may include onboard chip electronics 94. Chipelectronics 94 may include analog and/or digital circuitry. Eachsampling electrode of electrode array 54 may be in electrical contactwith chip electronics 94, indicated at 96. Each guard electrode 92 alsomay be in electrical contact with chip electronics 94, indicated at 98.(The horizontal/vertical positions of chip electronics 94 within chip 58are not shown accurately here.)

Chip electronics 94 also may be in electrical contact, indicated at 100,with optional device electronics 102 that are separate from chip 58.Device electronics 102 may include analog and/or digital circuitry, andmay have any suitable position in isolation device 90 relative to chip58, such as below, above, or laterally offset from chip 58. An optionalpower supply 104, such as a battery, may supply electrical power,indicated at 106, to chip electronics 94 and device electronics 102, oroff-device power (e.g., AC power from an electrical grid or an externalbattery) may be used as a power supply instead. Chip electronics 94 anddevice electronics 102 may form all of control circuit 72 of system 50(also see FIG. 1 ). Alternatively, a portion of control circuit 72 maybe formed by analog and/or digital circuitry that is separate fromisolation device 90. Accordingly, chip electronics 94 and/or deviceelectronics 102 may communicate with this external portion of controlcircuit 72, indicated respectively at 108 and 110, via respectivecommunication ports formed by isolation device 90.

Isolation device 90 may include a housing 112 to support, enclose,and/or protect chip 58 and other device components. Housing 112 may havea base 114 and a cover 116 received on the base. Cover 116 may becompletely removable or at least movable (e.g., pivotable or slidable)with respect to base 114. Counter electrode(s) 74 may be attached to anunderside of a body 118 of cover 116.

Isolation device 90 may form at least one vessel 120 to hold cells 52and electrolyte 80 (also see FIG. 1 ). Each vessel 120 may, for example,be a chamber, as depicted, including electrolyte-received space 81 andcell-receiving area 84 between chip 58 and counter electrode(s) 74. Inother examples, each vessel may be a channel (e.g., see Example 6). Atleast a portion of the floor of the vessel may be provided by chip 58.Side walls of vessel 120 may be formed by housing 112, such as by base114 thereof. At least a portion of the ceiling of vessel 120 may beformed by counter electrode(s) 74 and/or cover 116.

FIG. 3 is a flowchart listing exemplary steps that may be performed in amethod 130 of sampling material, such as nucleic acids, from cells usingan array of sampling electrodes. The steps listed in the flowchart ofFIG. 3 may be performed in any suitable order and combination, and maybe modified or supplemented as described elsewhere in the presentdisclosure, such as in Sections I, III, and IV.

Cells may be received on a chip including an array of samplingelectrodes, indicated at 131 a. A section of tissue including the cellsmay be received vertically above the electrode array, or isolated cellsmay be received vertically above the electrode array by flow of anelectrolyte in which the cells are located (also see Examples 5 and 6).

An electrical property may be measured for individual samplingelectrodes of the electrode array, indicated at 131 b. The electricalproperty may be electric current, voltage, resistance, or the like. Insome examples, a control circuit may apply a test voltage or a testcurrent to individual sampling electrodes and measure the current orvoltage resulting from the test voltage or current.

A lysis voltage may be applied to selected sampling electrodes for whichthe measured electrical property (e.g., electric current) satisfies atleast one condition, indicated at 131 c. For example, the lysis voltagefor a given sampling electrode may be applied if one or more values ormeasurements for the measured electrical property of the given samplingelectrode meet one or more criteria, which may be predefined before thesampling run begins. In some cases, a value for the measured electricalproperty of the given sampling electrode may be compared to a threshold,and the lysis voltage may be applied to the given sampling electrode ifthe value is less than (or greater than) the threshold.

The lysis voltage is applied between the given sampling electrode andthe counter electrode. This voltage has a magnitude and durationdesigned to lyse one or more cells located vertically above the givensampling electrode and/or a corresponding well (and/or aligned with thesampling electrode and/or well). The lysis voltage may be applied as asingle pulse, or two or more pulses, of elevated voltage for anysuitable total duration or duration for each pulse, such as less than10, 5, 2, or 1 second(s), among others. The lysis voltage may beelevated with respect to the test voltage applied in step 131 b, such asat least 2, 3, 4, or 5 times the test voltage. Accordingly, applicationof the lysis voltage may be described as applying a voltage spike to thegiven sampling electrode. The lysis voltage may have a predefinedprofile (magnitude and duration) for the sampling run, where the profileis chosen according to the type of cells/tissue being investigated, howthe cells and/or tissue is prepared, and/or the like.

Nucleic acids or other material released by step 131 c may be captured,indicated at 131 d. More specifically, an electrophoresis voltage may beapplied to the given sampling electrode, after application of the lysisvoltage. The electrophoresis voltage drives nucleic acidselectrophoretically to a capturing agent located at the given samplingelectrode and/or in the corresponding well, from one or more cellsaligned with (e.g., located vertically above or on) the given samplingelectrode and/or corresponding well. The electrophoresis voltage may (ormay not) be less than the lysis voltage applied to the given samplingelectrode, such as less than 50% or 30% of the lysis voltage. Theelectrophoresis voltage may be applied for any suitable amount of time,such as at least 1, 2, 5, or 10 seconds, and/or at least 2, 5, 10, or 20times the duration of the lysis voltage, among others.

Capture of nucleic acids may be achieved by any suitable non-covalentmolecular interactions, including hydrogen bonds, dipole-dipoleinteractions, and/or London dispersion forces, among others, and/or maybe achieved by covalently bonding the nucleic acids, or fragmentsthereof, to the particular tag located at the given sampling electrode.In some examples, the nucleic acids may be RNA molecules including apoly(A) tail (i.e., messenger RNAs), and capture may result fromhybridization of the poly(A) tail to a complementary sequence of thecapturing agent. In some examples, capture may be result frominteraction between DNA from a cell(s) and a DNA-binding agent, such asa DNA-binding protein. The DNA-binding agent may bind to DNA, such asdouble-stranded DNA, with or without sequence specificity. In someexamples, capture may include covalently attaching nucleic acids to atag that is immobilized at the given sampling electrode (e.g., seeExample 7).

Predicted-capture data for the array of sampling electrodes may beupdated and/or outputted, indicated at 131 e. The capture data may bepredicted based on application of the lysis voltage and/orelectrophoretic voltage. For example, capture of nucleic acids may bepredicted based on which sampling electrodes received the lysis voltageand/or the electrophoretic voltage. Alternatively, or in addition,capture may be predicted based on which sampling electrodes thatreceived the lysis voltage exhibited at least a threshold change in themeasured electrical property, when values of the electrical property arecompared before and after application of the lysis voltage. For example,if electric current is the electrical property measured, samplingelectrodes exhibiting at least a threshold increase in electric currentin response to application of the lysis voltage may be predicted to havelysed and released nucleic acids that were captured by the capturingagent at the corresponding sampling electrodes. The predicted-capturedata may represent a real time (or end of sampling run) estimate of thenumber, percentage, and/or locations at which cell lysis and capture oflysis-released nucleic acids are predicted to have occurred. Outputtingthe predicted-capture data may include displaying the capture data (witha display device) and/or printing the capture data with a printer.

Method 130 may return to step 131 b, indicated at 131 f, any suitablenumber of times for each sampling electrode of the electrode array,based in part on a decision whether to continue the sampling run,indicated at 131 g. The electrical property may be measured for eachsampling electrode only once before application of a lysis voltage tothe sampling electrode, or the electrical property may be monitored overtime, such as at regular intervals, before the lysis voltage is appliedto the sampling electrode. In some cases, the electrical property maycontinue to be monitored only for the sampling electrodes of theelectrode array that have not yet received the lysis voltage.Accordingly, all of the sampling electrodes may be monitored at first,and then fewer and fewer may be monitored over time as an increasingnumber of the sampling electrodes have received the lysis voltagefollowed by electrophoresis voltage. In some examples, the sampling runmay stop automatically, when at least a threshold number of samplingelectrodes have received the lysis voltage. In some examples, the usermay stop the sampling run based on predicted-capture data that has beenoutputted to the user. For example, the user may decide to abort thesampling run early if the predicted-capture data indicates the presenceof a technical problem.

Nucleic acids may be tagged with structurally different tags at aplurality of the sampling electrodes (and/or in corresponding wells),indicated at 131 h. One or more reagents for performing a taggingreaction, such as reagents for a reverse transcription reaction, may bereceived on the chip adjacent the electrode array. The nucleic acidstagged may be nucleic acids captured at step 131 d, complements of thesecaptured nucleic acids, or fragments of these captured nucleic acids.Each structurally different tag at one of the sampling electrodes of theplurality of sampling electrodes may include a different nucleotidesequence including an identifier. In some examples, the tag may be aprimer and tagging may be achieved by extending the primer to synthesizecomplements of captured RNA molecules at the sampling electrode, wherethe complements are covalently linked to the identifier. This primerextension may be catalyzed by a reverse transcriptase enzyme. In otherexamples, tagging may be achieved by covalently attaching the identifierto captured nucleic acids or fragments thereof.

A library of tagged nucleic acids may be prepared, indicated at 131 i.The library may represent tagged nucleic acids from a plurality of thesampling electrodes (and/or corresponding wells). Library constructionmay include releasing and pooling tagged nucleic acids from theplurality of sampling electrodes and/or corresponding wells, addinglinkers to tagged nucleic acids, transcribing tagged nucleic acids invitro with an RNA polymerase, amplifying tagged nucleic acids using PCR,or any combination thereof, among others. Members of the library may besequenced to obtain a nucleotide sequence from each member. Thenucleotide sequence may include a sequence of the identifier of one ofthe tags (or a perfect complement thereof) and a sequence from a nucleicacid released by a cell aligned with the tag, or from a complement ofthe nucleic acid.

III. EXAMPLES

The following examples describe selected exemplary aspects and featuresof the methods, systems, and devices of the present disclosure relatedto sampling/isolating nucleic acids from cells using an array ofsampling electrodes. These examples are intended for illustration onlyand should not limit the scope of the disclosure. The aspects andfeatures described in this section may be combined with one another andwith any combination of the aspects and features of the methods,systems, and devices disclosed elsewhere herein, such as in Sections I,II, and IV.

Example 1. Illustrative Electrical Measurements

This example describes illustrative electrical measurements that may berecorded with an exemplary embodiment of sampling system 50 byperformance of an exemplary implementation of sampling method 130 inwhich RNA released by cell lysis is captured by hybridization to acomplementary capturing agent; see FIGS. 4-6 (also see FIGS. 1-3 ).

FIG. 4 shows a pair of temporally aligned graphs presenting voltage andelectric current over the same time period for a single samplingelectrode of an electrode array of the sampling system. The samplingelectrode is located at the bottom of a well. The upper graph plots thevoltage applied to the sampling electrode over the time period, and thelower graph plots electric current that may be measured from theelectrode for the same time period.

Cell proximity testing may be performed during a testing phase 150 p ofthe time period. A positive test voltage 152 v may be applied to thesampling electrode (i.e., the sampling electrode is positive (the anode)and a counter electrode is negative (the cathode)). The test voltage maybe applied continuously or as one or a series of test pulses 154 t,which may be applied at regular intervals. Test pulses 154 t may producea temporally matching series of one or more measured current pulses 156c at the sampling electrode. Each measured current pulse 156 c may becompared with a threshold 158 t. Test pulses 154 t may be continueduntil a measured current pulse 156 c drops below threshold 158 t,indicated by an arrow at 160 d. This decrease in measured currentindicates an increase in electrical resistance between the samplingelectrode and the counter electrode, resulting from a cell being in veryclose proximity to the well over the sampling electrode. (The cell mayat least partially seal the mouth of the well with its plasma membrane(or wall).) The decrease in measured current also indicates that themembrane/wall of the cell should be easier to rupture upon applicationof a predefined lysis voltage, due to a reduced electrolyte/conductorcross section between the cell and the lip of the well. Furthermore, thedecrease in measured current indicates that nucleic acids from the cellcan be captured in the well once the cell is lysed.

A voltage spike 162 v may be applied to start a cell lysis phase 164 p,once the measured electric current drops below threshold 158 t. Voltagespike 162 v may reach a lysis voltage 166 v that is significantly higherthan the voltage of test pulses 154 t, such as at least 50%, 75%, 100%,150%, 200%, 500%, 1000%, or 2000% higher, among others. Moreover, themeasured current may increase disproportionately, as shown, relative tothe applied voltage, if cell lysis occurs, because the lysed cell offersless resistance to the flow of electric current. The drop in measuredelectric current below threshold 158 t may occur stochastically overtime and may be stochastically reversible, too. Accordingly, applicationof voltage spike 162 v may commence immediately once the measuredcurrent drops below threshold 158 t. The possible stochastic nature ofthis current drop means that different sampling electrodes of theelectrode array may exhibit a current drop below threshold 158 t atdifferent times relative to one another. Accordingly, the various phasesshown here may not be performed in parallel among sampling electrodes ofthe electrode array.

An RNA capture phase 168 p may be initiated at the end of lysis phase164 p. An electrophoresis voltage 170 v may be applied to the samplingelectrode, with the same voltage polarity as testing phase 150 p andlysis phase 164 p. Electrophoresis voltage 170 v drives nucleic acidsincluding RNA toward the sampling electrode because the nucleic acidsare negatively charged. The RNA can be captured by the capturing agentover the sampling electrode and/or in the well. The level ofelectrophoresis voltage 170 v may be significantly less than lysisvoltage 166 v, such as less than 50% of lysis voltage 166 v. Here,electrophoresis voltage 170 v is at the same level as test voltage 152v, but in other examples, electrophoresis voltage 170 v may be higher orlower than test voltage 152 v. Electrophoresis voltage 170 v may beapplied continuously or as a series of electrophoretic pulses, amongothers. Since electrophoresis voltage 170 v is applied at the same levelas test voltage 152 v in this example, the measured current after celllysis can be compared directly with the measured current before celllysis. Consistent with cell lysis having occurred, the measured currentduring RNA capture phase 168 p may remain fairly constant, with nofurther fluctuations below threshold 158 t.

A purification phase 172 p may be initiated after RNA capture phase 168p. A reverse voltage 174 r may be applied to the sampling electrode, todrive uncaptured nucleic acids away from the sampling electrode and outof the well. Reverse voltage 174 r has a polarity opposite that ofelectrophoresis voltage 170 v and may have any suitable magnituderelative to electrophoresis voltage 170 v. The absolute value of reversevoltage 174 r may be less than that of electrophoresis voltage 170 v, asshown here, or may be the same as or greater than that ofelectrophoresis voltage 170 v. The level of reverse voltage 174 r may beselected to balance the competing concerns of more efficient removal ofunbound nucleic acids using a higher reverse voltage against moreefficient retention of captured RNA during this phase using a lowerreverse voltage.

FIG. 5 shows a graph plotting illustrative electric current that may bemeasured from only a small subset (ten) of sampling electrodes of theelectrode array during performance of the exemplary sampling method. Thepre-lysis electric current for each electrode is represented by hatchedbars. Hatched bars without an adjacent “X” report the measured electriccurrent below threshold 158 t that triggered application of a lysisvoltage for the indicated sampling electrode. Hatched bars topped by an“X” report the lowest measured electric current for the indicatedsampling electrodes. Unfilled bars report the post-lysis electriccurrent measured after application of the lysis voltage, using the sametest voltage as for the pre-lysis current measurements. Samplingelectrodes for which the lysis voltage has not been applied do not havean unfilled bar accompanying the hatched bar because there is nopost-lysis current to report. Electrodes at positions 1, 2, 5, 7, and 8have received the lysis voltage and, after application of the lysisvoltage, exhibit a post-lysis current comparable to the pre-lysiscurrent of electrodes 3, 4, 6, 9, and 10.

FIG. 6 shows an exemplary predicted-capture map 176 m that may begenerated from the data of FIG. 5 in real time during performance of theexemplary sampling method. Predicted-capture map 176 m shows where RNAis predicted to have been captured according to electrode position,based on which sampling electrodes have received the lysis voltage. Morespecifically, sampling electrodes 1, 2, 5, 7, and 8 are represented bycross-hatched circles, and sampling electrodes 3, 4, 6, 9, and 10 arerepresented by unfilled circles. Since the sampling system may have anelectrode array of thousands of electrodes, the predicted-capture mapmay, for example, use color coding to indicate different percentages ofsampling electrodes that are predicted to have captured RNA, fordifferent sections of the electrode array.

Example 2. Electrode, Well, and Circuit Configurations

This example describes exemplary configurations for electrodes, wells,and associated energization/sensing circuits for the methods, systems,and devices of the present disclosure; see FIGS. 7, 8, 9A, 9B, and 10-14.

FIG. 7 shows a fragmentary portion of an exemplary embodiment (chip 258)of chip 58 (also see FIG. 1 ). Chip 258 has a rectangular electrodearray 254 of sampling electrodes 256, which may be regularly spacedalong each of two orthogonal axes, with the same spacing along bothaxes. The chip defines a corresponding, vertically aligned well array260 of wells 262, which are formed in a top surface 263 of chip 258.Each sampling electrode 256 of electrode array 254 is located at thebottom of a different well 262 of well array 260. Wells 262 may bedefined at least partially in a dielectric layer 265 of chip 258, suchthat the wells are electrically insulated from one another laterally.The center-to-center spacing between wells 262 may be greater than thediameter of wells 262. For example, in the depicted embodiment, the wellspacing is ten times the well diameter.

FIG. 8 shows a fragmentary portion of another exemplary embodiment (chip358) of chip 58 (also see FIG. 1 ). Chip 358 has a rectangular electrodearray 354 of sampling electrodes 356 similar to that of FIG. 7 , and awell array 360 of wells 362. Wells 362 are formed in a dielectric layer365, and in a top surface 363 of chip 358. Chip 358 also has a singleguard electrode 392 located on dielectric layer 365. Guard electrode 392may be formed of a plated conductive material. Guard electrode 392defines a plurality of openings 393 each aligned with (centered on) oneof sampling electrodes 356 and a corresponding well 362. Openings 393are larger in diameter than sampling electrodes 356 and wells 362. Thisdifference in diameter helps to electrically isolate each samplingelectrode 356 from guard electrode 392, to avoid interference betweenthese two types of electrodes. When wells 362 and guard electrode 392are projected vertically onto the same horizontal plane to form aprojection, guard electrode 392 encircles each sampling electrode 356and well 362 in the projection. Guard electrode 392 reduces or preventsconduction (i.e., electrical leakage) between wells 362. Accordingly,the guard electrode allows each well 362 to have an electrical signalthat is substantially unaffected by adjacent wells 362, which allows ahigher density of wells 362 (i.e., a smaller ratio of well spacing towell diameter). A guard voltage is applied to guard electrode 392 andmay be set to optimize the sampling of nucleic acids from a tissuesection, while reducing or preventing conduction between wells 362. Forexample, guard electrode 392 may be set to the same electric potentialas wells 362, or may be set to a different electric potential, such asthe electric potential at the bottom of a section of tissue above wells362.

FIG. 9A shows a fragmentary portion of yet another exemplary embodiment(chip 458) of chip 58 (also see FIG. 1 ). Chip 458 has a rectangularelectrode array 454 of sampling electrodes 456 similar to that of FIG. 8, and a well array 460 of wells 462 formed in a top surface 463 of thechip, in a dielectric layer 465 thereof. However, instead of having asingle guard electrode, as in chip 358 of FIG. 8 , chip 458 has a guardelectrode array 491 including a separate guard electrode 492 locatedaround each well 462 and defining an opening 493 aligned with the well.Like guard electrode 392 of FIG. 8 , guard electrode array 491 reducesor prevents conduction (i.e., electrical leakage) between wells 462.Accordingly, guard electrode array 491 allows each well 462 to have anelectrical signal that is unaffected by surrounding wells 462, whichallows a higher density of wells 462. An advantage of guard electrodearray 491, relative to single guard electrode 392 discussed above isthat guard electrodes 492 are independently controllable by the controlcircuit. Accordingly, a different guard voltage can be applied to therespective guard electrode 492 around each well 462. For example, therespective guard electrode may be set to the electric potential at thewell 462 or to another value such as the electric potential at thebottom of a tissue section located on chip 458. Guard electrode array491 allows the electric potential of each guard electrode 492 to beoptimized for spatial variation in the tissue section.

FIG. 9B shows a fragmentary portion of still another exemplaryembodiment (chip 558) of chip 58 (also see FIG. 1 ). Chip 558 has ahexagonal electrode array 554 of sampling electrodes 556, and acorresponding hexagonal well array 560 of wells 562 formed in a topsurface 563 of the chip, in a dielectric layer 565 thereof. Thishexagonal arrangement allows electrode array 554 and well array 560 tobe more compact. Chip 558 has a guard electrode array 591 ofindividually controllable guard electrodes 592 similar to guardelectrodes 492 described above for FIG. 9A, with each guard electrode592 located around a different well 562 and defining an opening 593centered on the well.

FIG. 10 shows a portion of an exemplary embodiment (isolation device690) of isolation device 90 including chip 358 of FIG. 8 (also see FIG.2 ). The portion of chip 358 shown includes one sampling electrode 356,one aligned well 362, and top surface 363 and dielectric layer 365 inwhich well 362 is formed.

Guard electrode 392 is also shown, including an opening 393 thereinaligned with well 362. Opening 393 may have an opening diameter that issubstantially larger than the diameter of the mouth (top) of well 362,such as an opening diameter that is at least 1.5, 2, or 3 times the welldiameter.

Copies of one tag 666 of a tag array of different tags (i.e., havingdifferent identifiers relative to one another) are located in well 362.The copies are attached to sampling electrode 356 via a porous layer 695located on the sampling electrode. Porous layer 695 immobilizes thecopies of tag 666 so that they remain connected to sampling electrode356 and in well 362 during the sampling run, even if a reverse voltageis applied to the sampling electrode. The porous layer permits contactof the electrolyte with sampling electrode 356, optionally whilepreventing nucleic acids from reaching the sampling electrode. Porouslayer 695 may be located in well 362, such as in a lower portionthereof, as shown here, and may, for example, be a hydrogel.

A substrate 697 is located under dielectric layer 365, optionally incontact with the dielectric layer. Substrate 697 may be formed of asemiconductor. Sampling electrode 356 may be located over, on, and/or insubstrate 697 and at the bottom of well 362. The sampling electrode may(or may not) form at least part of the floor of well 362, as shown here.

Isolation device 690 has a cover 716 including a body 718 and a counterelectrode 674, which is attached to an underside of the body. Anelectrolyte-receiving space 681 and a cell-receiving area 684 arelocated between counter electrode 674 and top surface 363 of chip 358. Atissue section may be received on top surface 363, namely, on guardelectrode 392 and dielectric layer 365, where the dielectric layer isnot covered by the guard electrode and defines the side wall portion ofwell 362. An electrolyte provides electrical contact between counterelectrode 674 and the tissue section, and between the tissue section andsampling electrode 356.

Electrical connections of the different types of electrodes 356, 392,and 674 to circuit portions of a control circuit are also shown. Counterelectrode 674 connects to voltage reference electronics at 699. Guardelectrode 392 connects to guard electronics at 701. Sampling electrode356 connects to sampling electronics at 703.

FIGS. 11-14 show circuit portions of exemplary control circuits. Eachcircuit portion enables setting one or more voltages and/or sensingcurrent.

FIG. 11 shows a circuit diagram of an exemplary circuit portion 805 ofan embodiment (control circuit 772) of control circuit 72 of samplingsystem 50 of FIG. 1 . Circuit portion 805 may be utilized to set thevoltage bias of a counter electrode, such as counter electrode 74 or 674(see FIGS. 1, 2, and 10 ), or of a guard electrode, such as guardelectrode 392 (see FIGS. 8 and 10 ).

Circuit portion 805 has an output 806 operably coupled to a counterelectrode or guard electrode (e.g., at 699 or 701 in FIG. 10 ). Anoperational amplifier 807 has a voltage reference set on the positiveinput (‘+’) by a voltage reference (Vref) 808, such as adigital-to-analog converter (DAC). The voltage set by voltage reference808 is driven to output 806 through an optional resistor 809. Output 806also provides negative feedback to the negative input (‘−’) ofoperational amplifier 807, such that output 806 matches the voltage setby voltage reference 808. Resistor 809, if included, serves as a currentsense resistor for the counter electrode or guard electrode, with senseelectronics not illustrated here. If included, resistor 809 is set to asmall value (e.g., 0.1Ω to 100Ω) with a value based on the maximumcurrent sampled. If current sensing is not needed, then resistor 809 maybe omitted from circuit portion 805 or set to zero ohms (0Ω).

FIG. 12 is a circuit diagram of an exemplary circuit portion 905 for anembodiment (control circuit 872) of control circuit 72 of samplingsystem 50 of FIG. 1 . Circuit portion 905 may be utilized to set thevoltage bias of segmented guard electrodes 492, 592 of FIGS. 9A and 9B.

Circuit portion 905 has an output 906 operably coupled to guardelectrodes 492 or 592. An operational amplifier 907 has a voltagereference set on the positive input (‘+’) by a voltage reference (Vref)908, such as a digital-to-analog converter (DAC). The voltage set byvoltage reference 908 is driven to output 906 through optional resistor909. Output 906 also provides negative feedback to negative input (‘−’)of operational amplifier 907, such that output 906 matches the voltageset by voltage reference 908. Resistor 909, if included, serves as acurrent sense resistor for segmented guard electrodes 492 or 592, withsense electronics not illustrated here. If included, resistor 909 is setto a small value (e.g., 0.1Ω to 100Ω) with a value based on the maximumcurrent sampled. If current sensing is not needed, then resistor 909 maybe omitted from the circuit or set to zero ohms (0Ω).

FIG. 13 is a circuit diagram of an exemplary transimpedance amplifiercircuit 1010 for an embodiment (control circuit 972) of control circuit72 of sampling system 50 of FIG. 1 . Amplifier circuit 1010 may beutilized to set the voltage bias and sense the current of samplingelectrodes 56, 256, 356, 456, or 556 of the sampling system (also seeFIGS. 1, 5-8, 9A, and 9B). An output 1011 is operably coupled to asampling electrode. Amplifier circuit 1010 both drives the voltage ofthe sampling electrode and senses the current thereof. An operationalamplifier 1012 has a voltage reference set on the positive input (‘+’)by a voltage reference (Vref) 1013, such as a digital-to-analogconverter (DAC). The voltage set by voltage reference 1013 is driven tooutput 1011 through the negative feedback to the negative input (‘−’) ofoperational amplifier 1012, such that output 1011 matches the voltageset by voltage reference 1013. A resistor 1014 serves as a current senseresistor for current sensing electronics 1015 to sense the current ofthe sampling electrode. Based on Ohm's law, the current of the samplingelectrode is determined by the difference between the output ofoperational amplifier 1012 and voltage reference 1013, divided by theresistance value of resistor 1014. Operational amplifier 1012 may have avery low input bias current, such that the current going into thenegative input (‘−’) of the operational amplifier is insignificantcompared to the sensed current of the sampling electrode.

FIG. 14 is a circuit diagram of an exemplary transimpedance amplifiercircuit 1110 for an embodiment (control circuit 1072) of control circuit72 of sampling system 50 of FIG. 1 . Amplifier circuit 1110 may beutilized to set the voltage bias and sense the current of samplingelectrodes 56, 256, 356, 456, or 556 of the sampling system (also seeFIGS. 1, 5-8, 9A, and 9B). An output 1111 is operably coupled to thesampling electrode. Amplifier circuit 1110 drives the voltage of thesampling electrode and senses current of the sampling electrode. Anoperational amplifier 1112 has a voltage reference set on the positiveinput (‘+’) thereof by a first voltage reference (Vref1) 1113, such as adigital-to-analog converter (DAC). The voltage set by first voltagereference 1113 is driven to output 1111 through negative feedback to thenegative input (‘−’) of operational amplifier 1112, such that output1111 matches the reference voltage set by first voltage reference 1113.A resistor 1114 serves as a current sense resistor for the samplingelectrode, where the current sensing electronics are comprised of acomparator 1116, a second voltage reference (Vref2) 1117, and digitalelectronics 1118. Based on Ohm's law, the current of the samplingelectrode is determined by the difference between the output ofoperational amplifier 1112 and the value of first voltage reference 1113divided by the resistance value of resistor 1114. Operational amplifier1112 may have a very low input bias current, such that the current goinginto the negative input (‘−’) of the operational amplifier isinsignificant compared to the sensed current of the sampling electrode.Second voltage reference 1117 is set to a value that causes comparator1116 to change state when the target current for the sampling electrodeis achieved.

The electronics of the sampling system may have any suitableconfiguration and distribution among system components. In someexamples, the sampling system may have an independent voltage referencefor each sampling electrode and independent sensing electronicsincluding a comparator, an ND, or both, such as the circuit depicted inFIG. 13 . This may be a complex system with an expensive implementation.The more expensive electronic components (voltage reference and A/D) maybe on independent equipment or a reusable chip carrier. The chip may bedisposable (single-use) or reusable. The chip carrier may use arechargeable battery or external source of power. In other embodiments,all of the electronics may be integrated onto the chip, which may bedisposable (single-use) or reusable. In some examples, the samplingsystem may include shared voltage references and multiplexers (Mux) forsampling electrodes, shared sensing electronics that are multiplexed(Mux) including a comparator, ND, or both, such as the circuit in FIG.13 . In still other embodiments, the sampling system may have sharedvoltage references and multiplexers (Mux) for the sampling electrodesand a comparator for each sampling electrode, with no expensive sensingelectronics such as an ND, as depicted in FIG. 14 .

Example 3. Isolation Device for Sampling Nucleic Acids from Cells

This example describes an exemplary embodiment, isolation device 1190,of isolation device 90 for the systems and methods of the presentdisclosure; see FIGS. 15-17 (also see FIG. 2 ).

Isolation device 1190 has a chip 1158 forming an array of samplingelectrodes 1156 aligned with an array of wells 1162 (see FIGS. 15 and 16). The size of each array (i.e., forty-two members) is small forclarity, namely, to make individual wells 1162 easily visible in thecontext of the entire isolation device 1190. Chip 1158 has a top surface1163 in which wells 1162 are formed, with each well having a side wallportion formed in a dielectric layer 1165, which may or may not form thelip of the well.

A housing 1212 of isolation device 1190 has side walls 1213 extendingabove chip 1158. Side walls 1213 serve as a barrier to lateral flow offluid and create a side wall portion of a vessel 1220. Top surface 1163of chip 1158 forms at least part of the floor of vessel 1220. Acell-receiving area 1181 is formed in vessel 1220, adjacent top surface1163. An electrolyte-receiving space 1181 is located over cell-receivingarea 1184.

A removable cover 1216 is operatively positionable overelectrolyte-receiving space 1181 (see FIG. 17 ). Cover 1216 is not shownin FIGS. 15 and 16 . Cover 1216 includes a counter electrode 1174attached to and supported by a body 1218, such that counter electrode1174 faces chip 1158 and each sampling electrode 1156 thereof.

Isolation device 1190 may have a series of electrical connectors, suchas pins 1221, attached to housing 1212 (see FIGS. 15 and 16 ). Theconnectors are used to electrically connect electronics of isolationdevice 1190 (such as chip electronics and/or other (non-chip) deviceelectronics) to other components of the control circuit and/or to asource of electrical power.

FIG. 17 shows a fragmentary sectional view of isolation device 1290taken through one sampling electrode 1256 and its corresponding well1262. Using at least one voltage source, an individually controllablevoltage can be applied to each sampling electrode 1256. In other words,the voltage is applied between counter electrode 1274, indicated byelectric potential V1, and the sampling electrode 1256, indicated byelectric potential V2, where the difference between V2 and V1 is theapplied voltage. Accordingly, application of a positive voltage tosampling electrode 1256 makes the sampling electrode a positiveelectrode or anode, while application of a negative voltage to samplingelectrode 1256 makes the sampling electrode a negative electrode orcathode.

Copies of a tag 1166 are supported by and attached to chip 1158 overelectrode 1156 in well 1162. More specifically, each copy of tag 1166 isconnected to electrode 1156 via a porous layer 1195 located on a topsurface of electrode 1156. Each copy of tag 1166 may be covalentlyattached to porous layer 1195 or associated with the porous layernoncovalently. The copies of each different tag 1166 may be synthesizedin situ on chip 1158, such as in situ on a respective porous layer 1195in well 1162, or may be synthesized separately and then deposited inwell 1162 of chip 1158. In other examples, copies of tag 1166 may besupported by chip 1158 but are not connected to the chip (see Example4).

Tag 1166 includes an identifier 1168 covalently linked to a capturingagent 1170, where each of identifier 1168 and capturing agent 1170includes a series of nucleotides. (“UMI” is an acronym for uniquemolecular identifier.) Here, capturing agent 1170 includes a series ofdeoxythymidine nucleotides (i.e., TTT . . . TTT) of any suitable length,such as at least 8, 10, 12, or more nucleotides. In other examples, thecapturing agent may include one or more deoxyuridine nucleotides, suchas an oligo(dU) sequence of at least 8, 10, 12, or more nucleotides. Inyet other examples, the capturing agent may include a combination ofdeoxythymidine and deoxyuridine residues. In still other examples, thecapturing agent may include a degenerate nucleotide sequence, (N)x,extending to the 3′-end of the tag, where N is a mix of A, C, G, and T(or U), and x is at least 3, 4, 5, 6, 7, or 8.

Chip 1158 has a series of layers each oriented parallel to top surface1163, as explained above in Example 2. These layers may include asubstrate 1197, dielectric layer 1165, and a cell adhesion layer 1222.Cell adhesion layer 1222 may be located over and/or on dielectric layer1165. The cell adhesion layer 1222 may include any suitable material toencourage overlying cells to adhere to top surface 1163 of chip 1158.For example, the cell adhesion layer may be a coating including apolypeptide, such as an Ig superfamily cell adhesion molecule, integrin,cadherin, selectin, polylysine, or the like.

Example 4. Isolation Device with Bead Array

This example describes an exemplary embodiment, isolation device 1290,of isolation device 90 for the systems and methods of the presentdisclosure; see FIG. 18 (also see FIG. 2 ).

Isolation device 1290 is structurally similar to isolation device 1190,except that each tag 1266 is supported by a chip 1258 but not connectedto the chip. Instead, copies of the tag 1266 are connected to a bead1323, which is located at least partially in a well 1262, optionallysupported on the top surface of electrode 1256. In the depictedembodiment, the diameter of bead 1323 is less than the depth of thewell, such that the bead is completely inside the well. In otherexamples, at least two beads connected to the same tag or different tagsmay be located in well 1262. In other examples, a portion of the beadmay protrude from the well. Beads of any suitable shape may be used.Beads may be advantageous because tags 1266 can be synthesized on thebeads separately from the chip, and then individual beads each carryinga different tag 1266 may be placed into different wells 1262. Moreover,synthesis of tags 1266 may be performed more easily on beads, such aswith a split-and-pool approach.

Example 5. Method of Sampling RNA from Cells of a Tissue Section

This example describes exemplary steps that may be performed, andconfigurations that may be generated, in a method of sampling RNA fromcells of a tissue section; see FIGS. 19-27 .

FIG. 19 show a flowchart listing steps of an exemplary method 1330 ofsampling cells of a tissue section for RNA. Method 1330 is an example ofmethod 130 of FIG. 3 described above in Section I. Steps 1330 a-1330 llisted in FIG. 19 may be performed in any suitable order andcombination, using any other suitable aspects of the systems and methodsof the present disclosure.

FIG. 20 shows a flowchart listing steps of an exemplarylysis/electrophoresis protocol 1331 that may be performed as step 1330 gwithin method 1330 of FIG. 19 . Steps 1331 a-1331 c listed in FIG. 20may be performed in any suitable order and combination, using any othersuitable aspects of the systems and methods of the present disclosure.

FIG. 21 shows a fragmentary sectional view of an exemplary configurationof an embodiment (isolation device 1390) of isolation device 90 (alsosee FIG. 2 ). The configuration shown here may represent step 1330 f ofmethod 1330 of FIG. 19 . A tissue section 1424 composed of a layer ofcells, including illustrative cells 1352 a, 1352 b, and 1352 c, issandwiched between an electrolyte gel 1380 and a surface of chip 1358 ofisolation device 1390. Tissue section 1424 adheres to the chip surface.Electrolyte gel 1380 allows application of uniform top-to-bottompressure and improves adhesion of tissue section 1424 to the surface ofchip 1358. A counter electrode 1374 may be in physical contact withelectrolyte gel 1380, or a layer of electrolyte liquid may be locatedbetween electrolyte gel 1380 and counter electrode 1374. A samplingelectrode 1356 and a corresponding well 1362 are aligned withillustrative cell 1352 b.

FIGS. 22-27 show exemplary configurations that may be produced withisolation device 1390 of FIG. 21 during performance of method 1330 ofFIG. 19 .

FIG. 22 shows isolation device 1390 and tissue section 1424 in the sameconfiguration as FIG. 21 , except with an electrolyte liquid 1380′,rather than an electrolyte gel, located between tissue section 1424 andcounter electrode 1374. Each cell 1352 a-1352 c contains nucleic acids1425, including messenger RNA molecules 1426 each having a poly(A) tail1427. Each cell also contains other nucleic acids 1428, such as RNAhaving no poly(A) tail or DNA (double-stranded or single-stranded). Cell1352 b is aligned with sampling electrode 1356 and located over themouth of the corresponding well 1362. The plasma membrane of cell 1352 bis in very close proximity to the top surface of chip 1358 around well1362, such that cell 1352 b reduces the electrical current measured atelectrode 1356 in response to application of a positive test voltage,indicated by “(+V)” and “(−V)” at sampling electrode 1356 and counterelectrode 1374, respectively. More specifically, the measured electriccurrent is lower than without cell 1352 b or lower than with a greaterseparation between cell 1352 b and the top surface of chip 1358 and isless than a threshold.

FIG. 23 schematically illustrates application of a lysis voltage tosampling electrode 1356, indicated as “(+V) High” below the samplingelectrode. The lysis voltage is rupturing the plasma membrane of cell1352 b, indicated at 1429, and nucleic acids 1428 are being releasedfrom cell 1352 b and traveling toward electrode 1356, indicated by amotion arrow at 1433. The greatest electric potential and inducedpressure is located near a narrowing of the conductor (electrolyte)between the lip of the well and cell 1352 b.

Electric potential dissipates rapidly with distance along the surface ofchip 1358 from well 1362. Accordingly, only the plasma membrane (and/orwall) of a cell(s) very near to well 1362 is ruptured.

FIG. 24 schematically illustrates application of an electrophoresisvoltage to sampling electrode 1356, indicated as “(+V) Medium” below thesampling electrode. The plasma membrane of cell 1352 b has been rupturedby the previously applied lysis voltage and does not significantlyobstruct travel of nucleic acids 1425 into well 1362 and toward samplingelectrode 1356, indicated by a set of motion arrows at 1433. Electricpotential dissipates quickly with distance along chip 1358 from well1362. Accordingly, only nucleic acids from cell 1352 b are drivenelectrophoretically into well 1362.

FIG. 25 schematically illustrates a configuration produced at the end ofthe electrophoresis phase, which is indicated by “(+V)” below samplingelectrode 1356. Messenger RNA molecules 1426 have entered well 1362 andare hybridized with copies of tag 1366. More specifically, the poly(A)tail 1427 of messenger RNA molecules 1426 is hybridized with anoligo(dT) capturing agent 1370 of tag 1366. Other nucleic acids 1428having no poly(A) tail have approached sampling electrode 1356 but arenot hybridized to capturing agent 1370.

FIG. 26 schematically illustrates a configuration produced during areverse electrophoresis phase. The reverse electrophoresis phase isdriven by reversing the polarity of the voltage applied to samplingelectrode 1356, as indicated by “(−V)” instead of “(+V)” below samplingelectrode 1356. Messenger RNA molecules 1426 remain captured byhybridization to immobilized capturing agent 1370 of tag 1366. Incontrast, unbound other nucleic acids 1428 are free to travel out ofwell 1362 in response to the reversed voltage, which helps to purify thecaptured messenger RNA molecules 1426 by separating them from othernucleic acids 1428.

FIG. 27 schematically illustrates a configuration produced after voltageapplication has been stopped and tissue section 1424 removed (comparewith FIG. 26 ). The top surface 1363 of chip 1358 has been washed, toremove cell debris and any unbound cell components. Areverse-transcription reaction mixture 1434 has been placed onto topsurface 1363 of chip 1358, such that reaction mixture 1434 enters eachwell 1362 of chip 1358. Reaction mixture 1434 includes a reversetranscriptase and dNTPs. The reverse transcriptase catalyzes extensionof hybridized copies of tag 1366 to form complementary DNA molecules,indicated at 1435, using tag 1366 as a primer and hybridized messengerRNA molecules 1426 as templates. After completion of the reversetranscription reaction, the resulting complementary DNA molecules can bereleased from chip 1358, pooled, and processed collectively to produce alibrary for sequencing. Library members originating from the same well1362, and thus the same cell or group of cells, can be identified afterpooling as containing the unique identifier 1368 for that well (or thecomplement of the unique identifier).

Example 6. Method and Device for Sampling Isolated Cells

This example describes an exemplary method and an exemplary embodiment(isolation device 1490) of isolation device 90 for sampling nucleicacids from single cells and/or small cell clusters that are notphysically connected to one another; see FIGS. 28-36 .

FIG. 28 shows an exploded view of isolation device 1490. A base 1535 ofisolation device 1490 includes a chip 1458 having an array of samplingelectrodes 1456 aligned with an array of wells 1462. Chip 1458 may haveany suitable combination of features and structures described elsewhereherein, including a top surface 1463 and a dielectric layer 1465.

A channel-forming assembly 1536 is attached to top surface 1463 of chip1458. Channel-forming assembly 1536 has a plurality of channels 1537provided by a corresponding number of capillary tubes 1538. Eachcapillary tube 1538 has a series of lateral openings 1539 for fluidcommunication with a series of wells 1462 of chip 1458. Capillary tube1538 form a fluid-tight seal with each well 1462 of a row of wells ofchip 1458.

FIGS. 29-34 show exemplary configurations produced by operation ofisolation device 1490 during performance of a sampling method accordingto FIG. 3 . Isolation device 1490 is shown as fragmentary and sectional,with the views taken through three wells 1462 arranged along one of therows of isolation device 1490 and the corresponding overlying capillarytube 1538. The configurations shown here conceptually correspond tothose shown above in FIGS. 22-27 in Example 5 for isolation device 1390and a tissue section instead of isolated cells.

FIG. 29 shows cells 1452 a-c in an electrolyte liquid 1480′ beingintroduced into channel 1537 by fluid flow, indicated at 1539 a. Cell1452 b is centered over sampling electrode 1456 b and a correspondingwell 1462 b, but cells 1452 a and 1452 c are not centered over samplingelectrodes 1456 a, 1456 c and wells 1462 a, 1462 c. A test voltage canbe applied to each sampling electrode 1456 a-c with the aid of a counterelectrode 1474 located in channel 1537. Measured electric currentproduced by the test voltage varies among sampling electrodes 1456 a-c,with sampling electrode 1456 b having a lower electric current thansampling electrodes 1456 a and 1456 c.

FIG. 30 shows a lysis voltage “(+V) High” being applied to samplingelectrode 1456 b in response to the lower electric current measured forthe sampling electrode. The membrane of cell 1452 b is being rupturedlocally, indicated at 1529.

FIG. 31 shows an electrophoresis voltage “(+V) Medium” being applied tosampling electrode 1456 b after application of the lysis voltage.Nucleic acids including a messenger RNA molecule 1526 and other nucleicacids 1528 are being driven into well 1462 b, and messenger RNA molecule1526 has hybridized with a tag 1466.

FIG. 32 shows well 1462 b after application of a reverse voltage to well1462 b, to remove nucleic acids not captured by capturing agent 1470 oftag 1466.

FIG. 33 shows the result of further flow of electrolyte liquid 1480along channel 1537, such that cells 1452 a and 1452 c having advanced(compare with FIG. 33 ). Cell 1452 c in now centered on well 1462 c,which produced a lower measured electric current for electrode 1456 c.As a result, a lysis voltage “(+V) High” is being applied to electrode1456 c, which produces local rupture of the cell membrane, indicated at1529.

FIG. 34 shows a configuration produced after mRNA molecules 1526 havebeen captured in each of wells 1462 b and 1462 c. Further flow ofelectrolyte liquid or a washing solution removes unbound cell componentsand non-lysed cells.

FIG. 35 shows an exemplary channel-forming member 1540 that can bebonded to the top surface of chip 1458 in place of channel-formingassembly 1536 (compare with FIG. 28 ). Channel-forming member 1540 maybe a sheet 1541 defining channels 1542 in a bottom surface of the sheet,with the channels having a spacing from one another matching the spacingbetween rows of wells 1462 of chip 1458.

FIG. 36 shows an exemplary chamber-forming member 1543 that can bebonded to the top surface of chip 1458 in place of channel-formingassembly 1536 (compare with FIG. 28 ). Chamber-forming member 1543 maybe a sheet defining (i) a recess forming the side walls and ceiling of achamber 1544, (ii) one or more inlet channels 1545 to carry cells inelectrolyte to chamber 1544, and (iii) one or more outlet channels 1546to carry cells in electrolyte from chamber 1544.

Example 7. DNA Capture and Tagging

This example describes an exemplary method, system, and isolation device1590 for sampling DNA from cells; see FIGS. 37 and 38 .

Isolation device 1590 may have any suitable combination of elements andfeatures described for isolation devices elsewhere herein. For example,the isolation device may include a chip 1558 having an array of samplingelectrodes, an aligned well array, and an aligned tag array (only onesampling electrode 1556, well 1562, and tag 1566 are shown here). Eachtag 1566 has an identifier 1568 that is unique to the tag array, asdescribed above for similar chips and isolation devices. The tag 1566also has at least one transposase recognition sequence, such as a pairof terminal inverted repeats (1648 a, 1648 b) (TIRs). Terminal invertedrepeats 1648 a, 1648 b are associated with a transposase 1649 (TnP),which is connected to sampling electrode 1556 via a porous layer 1595.

A capturing agent 1570 in the form of a DNA-binding protein 1651 (DBP)is connected to electrode 1556 via porous layer 1595. DNA-bindingprotein 1651 may bind DNA duplexes, such as DNA duplex 1653, withoutsubstantial sequence specificity. Before or after DNA duplex 1653 hasbeen bound by DNA-binding protein 1651, transposase 1649 covalentlyattaches, indicated at 1655, tag 1566 to a fragment 1657 of DNA duplex1653 via tagmentation (compare FIGS. 37 and 38 ). In other examples,DNA-binding protein 1651 may be omitted and transposase 1649 may act asa capturing agent 1570.

Example 8. Protein/Analyte Capture and Associated Tagging

This example describes an exemplary system and method for sampling a netpositively-charged or negatively-charged protein (or other chargedanalyte) from cells. A tag array may be formed in alignment with anelectrode array of sampling electrodes and an optional well array, asdescribed above. Copies of a capturing agent (i.e., a first bindingpartner), such as an antibody or other specific binding partner for theprotein/analyte, may be immobilized in alignment with each samplingelectrode of the electrode array (e.g., in a corresponding well alignedwith the sampling electrode). The protein/analyte may be released fromcells by electrical lysis, as described elsewhere herein, and driven byelectrophoresis of suitable polarity to the copies of the first bindingpartner aligned with corresponding sampling electrodes. The immobilizedfirst binding partner binds to (captures) the protein or other analyte,and then the electrophoresis polarity may be reversed to remove unboundcell components by reverse electrophoresis. After sampling has beencompleted at a desired number of sampling electrodes, further processingmay be performed in parallel with the captured protein/analyte. A secondbinding partner for the protein/analyte may be placed on the tag array.The second binding partner may be labeled with an oligonucleotide. Thefirst and second binding partners may be configured to bind theprotein/analyte non-competitively, such that both can be bound to thesame copies of the protein/analyte at the same time. The proximity ofunique tags and the oligonucleotide to one another at individualpositions of the array then may be utilized to performproximity-dependent ligation or extension. In proximity-dependentligation, the tags and the oligonucleotide are ligated to one another,optionally with the aid of a splint oligo. In proximity-dependentextension, the tags and/or the oligonucleotide are extended whilehybridized to one another. With either approach, the resultant ligationproducts or extension products may be pooled and used to prepare alibrary for sequencing.

IV. Selected Aspects

This section describes selected aspects of the present disclosure as aseries of indexed paragraphs.

-   -   A1. A system for sampling material from cells, the system        comprising: (a) a chip including an electrode array of sampling        electrodes arranged along a surface of the chip, wherein a        cell-receiving area is located adjacent the surface of the        chip; (b) a tag array supported by the chip and aligned with the        electrode array, the tag array being composed of tags, each tag        of the tag array including an identifier that is unique to the        tag within the tag array; and (c) a control circuit configured        to apply an individually controllable voltage to each sampling        electrode of the electrode array and measure an electrical        property of the sampling electrode.    -   A2. The system of paragraph A1, wherein each tag of the tag        array is a primer configured to hybridize to a poly(A) tail of        RNA molecules.    -   A3. The system of paragraph A2, wherein the primer includes an        oligo(dT) sequence of at least 3, 5, 7, or 10 consecutive        deoxythymidine nucleotides at a 3′-end of the primer.    -   A4. The system of paragraph A2, wherein the primer includes a        degenerate nucleotide sequence at a 3′-end of the primer.    -   A5. The system of paragraph A1, wherein copies of a capturing        agent for nucleic acids or a protein are supported by the chip        and aligned with each tag of the tag array, and wherein the        capturing agent is not provided by the tag array, and wherein,        optionally, the capturing agent is a specific binding partner        (e.g., an antibody) for the protein.    -   A6. The system of paragraph A1, wherein the capturing agent        includes a transposase.    -   A7. The system of paragraph A6, wherein the transposase is        complexed with each tag of the tag array.    -   A8. The system of any of paragraphs A5 to A7, wherein the        capturing agent includes a DNA-binding protein.    -   A9. The system of paragraph A8, wherein the DNA-binding protein        binds double-stranded DNA.    -   A10. The system of any of paragraphs A1 to A9, wherein each tag        of the tag array is connected to the chip.    -   A11. The system of paragraph A10, wherein each tag is attached        to a porous layer located on a sampling electrode of the        electrode array.    -   A12. The system of any of paragraphs A1 to A9, wherein each tag        of the tag array is attached to a bead that is supported by the        chip and located at least partially in a well defined by the        chip, and wherein the bead is removable from the well.    -   A13. The system of any of paragraphs A1 to A12, wherein a well        array of wells is formed in the surface of the chip, wherein        each sampling electrode of the electrode array is located at the        bottom of a different well of the well array, and wherein,        optionally, each tag of the tag array is located in one of the        wells of the well array.    -   A14. The system of paragraph A13, wherein each well of the well        array has a diameter of less than 100, 50, 20, 10, or 5        micrometers.    -   A15. The system of paragraph A13 or A14, wherein each well of        the well array has a depth of less than 100, 50, 20, 10, or 5        micrometers.    -   A16. The system of any of paragraphs A13 to A15, wherein the        well array has an average center-to-center spacing between the        wells that is at least twice an average diameter of the wells.    -   A17. The system of any of paragraphs A13 to A16, wherein the        well array has an average center-to-center spacing between the        wells that less than 20, 10, or 5 times an average diameter of        the wells.    -   A18. The system of any of paragraphs A13 to A17, wherein the        chip includes a dielectric layer, and wherein a side wall        portion of each well is formed by the dielectric layer.    -   A19. The system of any of paragraphs A1 to A18, wherein the        electrode array includes at least 1,000 electrodes.    -   A20. The system of any of paragraphs A1 to A19, wherein the        electrode array is a planar array.    -   A21. The system of any of paragraphs A1 to A20, wherein the chip        includes a substrate composed of a semiconductor, and wherein,        optionally, the chip includes an integrated circuit, and        wherein, optionally, the integrated circuit includes digital        electronics.    -   A22. The system of any of paragraphs A1 to A21, further        comprising one or more counter electrodes, wherein the control        circuit is configured to apply the individual controllable        voltage between the one or more counter electrodes and each        sampling electrode of the electrode array.    -   A23. The system of paragraph A22, where the one or more counter        electrodes are configured to face the electrode array.    -   A24. The system of paragraph A22 or A23, wherein the one or more        counter electrodes are a single counter electrode.    -   A25. The system of any of paragraphs A22 to A24, wherein the        cell-receiving area is located intermediate the electrode array        and the one or more counter electrodes when the one or more        counter electrodes are operatively positioned with respect to        the electrode array, and/or wherein the one or more counter        electrodes are configured to be movable with respect to the        electrode array, to permit a tissue section to be placed in the        cell-receiving array without interference from the one or more        counter electrodes.    -   A26. The system of any of paragraphs A22 to A25, further        comprising an electrolyte-receiving space between the        cell-receiving area and the one or more counter electrodes when        the one or more counter electrodes are operatively positioned        with respect to the electrode array.    -   A27. The system of paragraph A26, further comprising an        electrolyte gel configured to be disposed in the        electrolyte-receiving space.    -   A28. The system of paragraph A27, wherein the electrolyte gel is        a sheet.    -   A29. The system of any of paragraphs A1 to A28, wherein the chip        includes one or more guard electrodes that are energizable by        the control circuit and located along the surface of the chip        intermediate sampling electrodes of the electrode array.    -   A30. The system of paragraph A29, further comprising one or more        counter electrodes that are shared by the electrode array and        the one or more guard electrodes.    -   A31. The system of paragraph A29 or A30, wherein the electrode        array defines a plane, and wherein the one or more guard        electrodes are offset from the plane.    -   A32 The system of any of paragraphs A1 to A31, wherein the chip        forms at least a portion of a floor of a vessel that includes        the cell-receiving area.    -   A33. The system of paragraph A32, further comprising a housing        that forms side walls of the vessel.    -   A34. The system of paragraph A33, wherein the housing includes a        cover for the vessel, wherein one or more counter electrodes for        the electrode array form at least part of the cover.    -   A35. The system of paragraph A34, wherein the cover is movable        with respect to a base of the housing.    -   A36. The system of any of paragraphs A1 to A31, wherein the        cell-receiving area includes at least one channel disposed in        fluid communication with a plurality of sampling electrodes of        the electrode array.    -   A37. The system of any of paragraphs A1 to A36, wherein the        control circuit is configured to apply a lysis voltage to each        sampling electrode of the electrode array, to lyse a cell        located in the cell-receiving area and aligned with the sampling        electrode, if the electrical property measured for the sampling        electrode meets one or more criteria.    -   A38. The system of paragraph A37, wherein the electrical        property is electric current, and wherein the control circuit is        configured to apply the lysis voltage to the sampling electrode        if the electric current measured for the sampling electrode is        below a threshold.    -   A39. The system of paragraph A38, wherein the control circuit is        configured to apply a test voltage to each sampling electrode of        the electrode array and measure an electric current produced by        the test voltage, and wherein the lysis voltage is greater than        the test voltage.    -   A40. The system of paragraph A39, wherein the lysis voltage        includes a voltage spike that is at least twice the test        voltage, and/or wherein the control circuit is configured to        measure the electrical property again for the sampling electrode        after the lysis voltage has been applied.    -   A41. The system of any of paragraphs A37 to A40, wherein the        control circuit is configured to apply an electrophoresis        voltage to the sampling electrode after applying the lysis        voltage, to drive nucleic acids toward the sampling electrode        for capture by a tag of the tag array (or for capture by a        distinct capturing agent that is not provided by the tag array).    -   A42. The system of paragraph A41, wherein the control circuit is        configured to apply a reverse voltage to the sampling electrode        after application of the electrophoresis voltage, to remove        nucleic acids not captured by the tag of the tag array or the        distinct capturing agent.    -   A43. The system of any of paragraphs A37 to A42, wherein the        control circuit is configured to generate data representing a        percentage, number, and/or map of sampling electrodes of the        electrode array at which capture of material (such as nucleic        acids) from lysed cells is predicted to have occurred.    -   A44. The system of paragraph 43, wherein the control circuit is        configured to generate the data based on (a) the electrical        property measured for each sampling electrode, (b) which or how        many sampling electrodes of the electrode array have received a        lysis voltage, (c) which or how many sampling electrodes of the        electrode array have received an electrophoresis voltage, or (d)        any combination thereof.    -   A45. The system of paragraph A44, wherein the control circuit is        configured to output the data as the control circuit continues        to measure the electrical property for sampling electrodes of        the electrode array at which capture of nucleic acids is        predicted not to have occurred.    -   A46. The system of paragraph A44 or A45, wherein the control        circuit is configured to control display of the data to a user.    -   A47. The system of any of paragraphs A1 to 46, wherein the        surface is a top surface of the chip, and/or wherein the surface        is planar, and/or wherein the surface forms a surface of the        chip in which wells are formed and each sampling electrode is        located at the bottom of a different one of the wells, and/or        wherein each tag of the tag array is located in one of the wells        of the well array.    -   A48. The method of any of paragraphs A1 to A47, further        comprising any limitation or combination of limitations of        paragraphs B1 to B19, C1, and D1 to D3.    -   B1. A method of sampling nucleic acid material from cells, the        method comprising: (a) receiving a plurality of cells on a        surface of a chip, the chip including an electrode array of        sampling electrodes arranged along the surface; (b) measuring an        electrical property for each sampling electrode of the electrode        array; (c) applying a lysis voltage to the sampling electrode if        the measured electrical property for the sampling electrode        meets one or more criteria; and (d) applying an electrophoresis        voltage to the sampling electrode, if the lysis voltage was        applied to the sampling electrode, to drive nucleic acids, if        any, that were released from one of the cells by applying the        lysis voltage, toward the sampling electrode.    -   B2. The method of paragraph B1, wherein the chip includes a tag        array of tags aligned with the electrode array, wherein each tag        includes an identifier that is unique within the tag array, and        wherein applying an electrophoresis voltage includes driving the        nucleic acids, if any, to copies of a tag of the tag array that        is aligned with the sampling electrode.    -   B3. The method of paragraph B2, wherein the tag is configured to        hybridize with RNA molecules of the nucleic acids, to capture        the RNA molecules.    -   B4. The method of any of paragraphs B1 to B3, wherein receiving        a plurality of cells includes receiving a tissue section        including the plurality of cells.    -   B5. The method of paragraph B4, further comprising receiving an        electrolyte gel on the tissue section.    -   B6. The method of any of paragraphs B1 to B3, wherein receiving        a plurality of cells including driving cells by fluid flow into        a cell-receiving area located adjacent the surface of the chip.    -   B7. The method of any of paragraphs B1 to B6, wherein receiving        a plurality of cells includes receiving a plurality of cells on        a well array of wells aligned with the electrode array.    -   B8. The method of paragraph B7, wherein receiving a plurality of        cells includes receiving a plurality of cells in respective        alignment with each well of a plurality of wells of the well        array.    -   B9. The method of any of paragraphs B1 to B8, wherein measuring        an electrical property includes applying a test voltage to each        sampling electrode of the electrode array and measuring an        electric current produced by the test voltage.    -   B10. The method of paragraph B9, wherein applying a lysis        voltage includes applying a lysis voltage to the sampling        electrode if the electric current measured for the sampling        electrode is below a threshold.    -   B11. The method of any of paragraphs B1 to B10, further        comprising capturing nucleic acids with a capturing agent over        each sampling electrode of a plurality of sampling electrodes of        the electrode array.    -   B12. The method of paragraph B11, wherein capturing nucleic        acids includes capturing RNA molecules of the nucleic acids by        hybridization with the capturing agent.    -   B13. The method of any of paragraphs B1 to B12, wherein a tag        array is aligned with the electrode array, wherein the tag array        is composed of tags, wherein each tag has an identifier that is        unique within the tag array, and wherein applying an        electrophoresis voltage includes driving nucleic acids from the        one cell, if any, that was lysed by the lysis potential, to a        tag of the tag array that is aligned with the one cell.    -   B14. The method of paragraph B13, further comprising driving        nucleic acids from two or more cells of the plurality of cells        to a corresponding number of different tags of the tag array.    -   B15. The method of paragraph B14, further comprising covalently        attaching each of the different tags to DNA from a different one        of the two or more cells.    -   B16. The method of any paragraphs B1 to B15, further comprising        outputting data representing a percentage, number, and/or map of        sampling electrodes of the electrode array at which capture of        nucleic acids from lysed cells is predicted to have occurred.    -   B17. The method of paragraph B16, wherein outputting data        includes displaying the data.    -   B18. The method of paragraph B17, further comprising displaying        the data as the control circuit continues to measure the        electrical property for sampling electrodes of the electrode        array at which a lysis voltage has not been applied.    -   B19. The method of any of paragraphs B1 to B18, further        comprising preparing a library for sequencing, the library        including nucleic acids captured over each sampling electrode of        a plurality of sampling electrodes of the electrode array, or        the library including fragments or complements of such captured        nucleic acids.    -   B20. The method of any of paragraphs B1 to B19, further        comprising any limitation or combination of limitations of        paragraphs A1 to A47.    -   C1. A method of sampling nucleic acid material from cells, the        method comprising: (a) receiving a tissue section on a surface        of a chip, the chip including an electrode array of sampling        electrodes arranged along the surface; (b) measuring an        electrical property for each sampling electrode of the electrode        array; (c) applying a lysis voltage to the sampling electrode if        the measured electrical property for the sampling electrode        meets one or more criteria; and (d) applying an electrophoresis        voltage to the sampling electrode to drive nucleic acids, if        any, that were released from a cell of the tissue section by the        lysis voltage, to copies of a capturing agent for the nucleic        acids.    -   D1. A method of sampling nucleic acid material from a tissue        section, the method comprising: (a) selecting a chip including        an electrode array of sampling electrodes arranged along a        surface of the chip, the chip supporting a primer array aligned        with the electrode array, the primer array being composed of        primers, each primer being configured to hybridize to a poly(A)        tail of RNA and including an identifier that is unique to the        primer within the primer array; (b) receiving the tissue section        on the surface of the chip; (c) measuring an electrical property        for each sampling electrode of the electrode array; (d) applying        a lysis voltage to the sampling electrode if the measured        electrical property for the sampling electrode meets one or more        criteria; and (e) capturing RNA molecules from the tissue        section with each primer of a plurality of different primers of        the primer array.    -   D2. The method of paragraph D1, the method further comprising        applying an electrophoresis voltage to the sampling electrode to        drive nucleic acids, if any, that were released from a cell of        the tissue section by the lysis voltage, to a primer of the        primer array that is aligned with the sampling electrode.    -   D3. The method of paragraph D1 or D2, further comprising any        limitation or combination of limitations of paragraphs Al to        A48, B1 to B20, and C1.

The term “exemplary” as used in the present disclosure, means“illustrative” or “serving as an example.” Similarly, the term“exemplify” means “to illustrate by giving an example.” Neither termimplies desirability or superiority.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.Further, ordinal indicators, such as first, second, or third, foridentified elements are used to distinguish between the elements, and donot indicate a particular position or order of such elements, unlessotherwise specifically stated.

We claim:
 1. A method of sampling nucleic acid material from tissue, themethod comprising: receiving a tissue section on a surface of a chip,the chip including an electrode array of sampling electrodes arrangedalong the surface; measuring an electrical property for each samplingelectrode of the electrode array; applying a lysis voltage to thesampling electrode if the measured electrical property for the samplingelectrode meets one or more criteria; and applying an electrophoresisvoltage to the sampling electrode, if the lysis voltage was applied tothe sampling electrode, to drive nucleic acids, if any, that werereleased from one of the cells of the tissue section by applying thelysis voltage, toward the sampling electrode.
 2. The method of claim 1,wherein the chip includes a tag array of tags aligned with the electrodearray, wherein each tag includes an identifier that is unique within thetag array, and wherein applying an electrophoresis voltage includesdriving the nucleic acids, if any, to copies of a tag of the tag arraythat is aligned with the sampling electrode.
 3. The method of claim 2,wherein the tag is configured to hybridize with RNA molecules of thenucleic acids, to capture the RNA molecules.
 4. The method of claim 1,further comprising receiving an electrolyte gel on the tissue section.5. The method of claim 1, further comprising capturing nucleic acidswith a capturing agent over each sampling electrode of a plurality ofsampling electrodes of the electrode array, wherein capturing nucleicacids includes capturing RNA molecules of the nucleic acids byhybridization with the capturing agent.
 6. The method of claim 1,wherein a tag array is aligned with the electrode array, wherein the tagarray is composed of tags, wherein each tag has an identifier that isunique within the tag array, and wherein applying an electrophoresisvoltage includes driving nucleic acids from the one cell, if any, thatwas lysed by the lysis potential, to a tag of the tag array that isaligned with the one cell.
 7. The method of claim 1, further comprisingoutputting data representing a percentage, number, and/or map ofsampling electrodes of the electrode array at which capture of nucleicacids from lysed cells is predicted to have occurred.
 8. A method ofsampling nucleic acid material from cells, the method comprising:receiving a tissue section on a surface of a chip, the chip including anelectrode array of sampling electrodes arranged along the surface;measuring an electrical property for each sampling electrode of theelectrode array; applying a lysis voltage to the sampling electrode ifthe measured electrical property for the sampling electrode meets one ormore predefined criteria; and applying an electrophoresis voltage to thesampling electrode to drive nucleic acids, if any, that were releasedfrom a cell of the tissue section by the lysis voltage, to copies of acapturing agent for the nucleic acids.